Development of complex neuronal morphology and accurate neural connections requires tightly controlled axon outgrowth and responses to guidance signals in the cell environment. Precise subcellular localization of guidance cue receptors and localized signaling to the cytoskeleton are essential for axon guidance. Proteins are targeted to specific cell locations by complex membrane trafficking and axonal transport systems. Neurons are particularly dependent on diverse and high fidelity protein trafficking because of their highly polarized and complex structure. Defects in protein trafficking and transport underlie multiple human developmental and neurodegenerative diseases, including Alzheimer's disease, Charcot-Marie-Tooth, and Niemann Pick disease. Despite their importance, the mechanisms regulating these critical processes in neurons are poorly understood. A major challenge to the field and the long term goal of this project is to understand how protein localization and cytoskeletal dynamics are controlled as neurons develop in their natural environment, where they must integrate multiple extracellular cues. We established a model in which we can image dynamics of neuronal cargo transport, protein localization, and cytoskeletal changes in the intact zebrafish embryo. Vertebrate sensory neurons must extend distinct central and peripheral axons to form the sensory circuit. We found that these axons show distinct responses to axon guidance cues. Moreover, we discovered roles for endosomal trafficking and the kinesin adaptor Calsyntenin-1 (Clstn-1) in sensory axon guidance. In Aim 1 we propose to determine how Clstn-1 regulates endosome transport routes to different axon compartments. In Aim 2 we will investigate mechanisms regulating specific localization of receptors for Neurotrophin-3 and Semaphorin3d. We will test the hypothesis that Clstn-1 and another class of kinesin adaptors, the Collapsin response mediator proteins (CRMPs) function to target receptors to specific axon compartments. In Aim 3 we will determine how Clstn-1, CRMPs, Sema3d and Neurotrophin-3 converge to regulate localized cytoskeletal dynamics. Our unique model allows us to connect the molecular events of axonal transport, guidance receptor localization and cytoskeletal changes to specific axon guidance decisions at the time and place they naturally occur. Elucidation of the molecular signals regulating sensory axon growth, guidance, and protein trafficking is critical for understanding neurodegenerative disorders, neuropathic pain disorders and the conditions under which regeneration after axon injury can occur. Our experiments will uncover such mechanisms and thus may help to identify molecular targets for disease treatment.